RAS Association Domain Family Member 4 (RASSF4): A New Potent Tumor Suppressor in Multiple Myeloma

Multiple myeloma (MM) is a genetic highly heterogeneous plasma cell malignancy. In MM, the RAS/MAPK pathway is the most frequently mutated pathway, leading to aberrant MEK/ERK and PI3K/Akt signaling and thus stimulating MM cell growth and survival. Increasing evidence indicates that next to their classical oncogenic effects, RAS proteins can also induce growth inhibitory effects and apoptosis. These tumor suppressive effects are mainly mediated through the Ras-Association Domain Family (RASSF), a group of 10 RAS effector proteins (RASSF1-10) and numerous isoforms. Based on the localization of the Ras-association (RA) domain this family is further subdivided into two groups. RASSF1 to 6 harbor a C-terminal RA domain and are termed the classical group, while RASSF7-10 contain a N-terminal RA domain and are referred to as non-classical RASSF proteins. The classical RASSF members also carry a SARAH domain on the C-terminus, allowing the RASSF proteins to bind each other and the mammalian sterile 20-like kinases MST and MST-2. Currently, no data about the biological role of RASSF proteins in the pathogenesis of MM is available.

Here, we first investigated the expression of the classical RASSF proteins RASSF1-6 in MM using publically available gene expression profiling data of 2 independent newly diagnosed patient cohorts (namely the Heidelberg-Montpellier and the UAMS-TT2 cohort) and 1 cohort of relapsed patients (Mulligan-cohort). We report that RASSF4 was the only classical RASSF protein that was consistently found downregulated in MM cells compared to normal bone marrow plasma cells, correlating with a bad prognosis in all cohorts. Treatment with epigenetic modulating agents including histone deacetylase inhibitors (HDACi) and DNA methyltransferases inhibitors significantly increased RASSF4 expression both in vitro and in vivo, indicating that RASSF4 downregulation in MM is due to epigenetic silencing. Forced RASSF4 expression induced a strong G2-phase arrest and caspase-3 mediated apoptosis in human MM cell lines and strongly reduced the viability of primary CD138+ MM cells. Moreover, RASSF overexpression significantly reduced in vivotumor growth. In addition, we showed RASSF4-MST1 binding and the activation of the downstream signaling pathways JNK/Jun, p38 and p53. RNA sequencing and gene set enrichment analysis following forced RASSF4 expression furthermore revealed downregulation of genes involved in protein metabolism, the unfolded protein response and translation. RASSF4 overexpression also sensitized MM cells to the proteasome inhibitor bortezomib, the specific MEK1/2 inhibitor trametinib and the ROS inducer Prima-1Met. Consequently, combining trametinib with HDACi, e.g. panobinostat, resulted in very strong synergistic anti-MM effects.

In conclusion, we identified RASSF4 as a new potent tumor suppressor in MM that is epigenetically silenced in malignant plasma cells and provide a rationale for testing novel therapeutic strategies enhancing RASSF4 expression (using HDACi or gene therapy) in combination with MEK/ERK inhibitors and bortezomib.